Considering that more than 50% of cancer occurs in the elderly and the immune system of the old is associated with a dramatic reduction in responsiveness, studies from our group and others indicate that immunotherapies that are effective in the young are not necessarily effective in the old. To effectively develop an immunotherapeutic strategy and be able to translate this approach for the treatment of cancers in the old, it is necessary to: 1) use relevant models that closely reflect those of cancer patients where self-tolerance and aging are present simultaneously;and 2) identify and understand the intrinsic defects of the old immune system. We have developed a tumor model by crossing the FVB-Her-2/neu mice with HLA-A2 mice (A2xneu). These animals are tolerant to Her-2/neu antigens and spontaneous tumors appear when they are 22-27 months old. Therefore, the A2xneu mouse model represents a unique model where aging and tolerance are present simultaneously allowing us to evaluate the immune and antitumor responses in an old-tolerant environment. Our preliminary results indicate that treatment with intratumoral injections of CpG-ODN plus peptide vaccination and depletion of Tregs induce an immune response that reject the primary tumors in 100% of young A2xneu mice and all young animals developed a protective memory response. In contrast, old A2xneu mice only delay the tumor growth. Although we were able to overcome tolerance in young A2xneu mice, these results suggest that other deficiencies may exist in old A2xneu mice preventing the optimal activation of an immune response in these animals. Analysis of the function of young and old dendritic cells (DCs) indicates that old DCs were suboptimally activated after CpG-ODN stimulation when compared to young DCs. However, the function of old DCs could be restored if old DCs were stimulated with CpG-ODN plus anti-CD40. Additionally, our results indicate that a higher number of old naive CD8+ T cells express B7-H1. Since B7-H1 negatively regulates T cell responses, we hypothesized that the expression of B7-H1 in old CD8+ T cells could restrict or diminish the immune function of these cells. Indeed, our results indicate that blockade of B7- H1 in old CD8+ T cells restore the function of these cells. Based on these results, we hypothesized that the combination of CpG-ODN, anti-CD40, Tregs depletion and anti-B7-H1, will induce stronger antitumor immune responses capable of controlling the tumor growth in old tolerant A2xneu mice. Preliminary results indicate that this is the case. The incorporation of anti-CD40 and anti-B7-H1 in our vaccination protocol is important because: 1) the addition of anti-CD40 into the CpG-ODN vaccination protocol will restore the function of DC resulting in stronger immune responses;and 2) blockade of B7-H1 will restore the function of old CD8 T cells permitting the optimal activation and expansion of these cells. Aim 1 will evaluate how blockade of B7-H1 restore the function of old CD8 T cells and how the combination of CpG+anti-CD40 enhances the function of old DCs. Aim 2 will optimize and evaluate the biological effects on the immune responses following immunization with CpG-ODN, anti-CD40, Tregs depletion and anti-B7-H1 in old tolerant hosts (old A2xneu mice). Aim 3 will evaluate the antitumor responses using the optimal immunotherapy condition. The information of these studies will reveal new strategies for controlling and manipulating the immune system to develop more effective cancer-immunotherapies in old tolerant hosts.